Electropipettor and compensation means for electrophoretic bias

ABSTRACT

The present invention provides for techniques for transporting materials using electrokinetic forces through the channels of a microfluidic system. The subject materials are transported in regions of high ionic concentration, next to spacer material regions of high ionic concentration, which are separated by spacer material regions of low ionic concentration. Such arrangements allow the materials to remain localized for the transport transit time to avoid mixing of the materials. Using these techniques, an electropipettor which is compatible with the microfluidic system is created so that materials can be easily introduced into the microfluidic system. The present invention also compensates for electrophoretic bias as materials are transported through the channels of the microfluidic system by splitting a channel into portions with positive and negative surface charges and a third electrode between the two portions, or by diffusion of the electrophoresing materials after transport along a channel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSerial No. 08/760,446, filed Dec. 6, 1996, and a continuation of andclaims the benefit of U.S. patent application Ser. No. 08/883,638, filedJun. 26, 1997, now U.S. Pat. No. 5,958,203 which is acontinuation-in-part of U.S. patent application Ser. No. 08/760,446,filed Dec. 6, 1996, now U.S. Pat. No. 5,880,071, which is acontinuation-in-part of U.S. patent application Ser. No. 08/671,986,filed Jun. 28, 1996, now U.S. Pat. No. 5,779,868, all of which areincorporated herein by reference in their entirety for all purposes,which is a continuation-in-part of U.S. patent application Ser. No.08/671,986, filed June 28, 1996, all of which are incorporated herein byreference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

There has been a growing interest in the manufacture and use ofmicrofluidic systems for the acquisition of chemical and biochemicalinformation. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc., are being used in the fabrication of these microfluidic systems.The term, "microfluidic", refers to a system or device having channelsand chambers which are generally fabricated at the micron or submicronscale, e.g., having at least one cross-sectional dimension in the rangeof from about 0.1 μm to about 500 μm. Early discussions of the use ofplanar chip technology for the fabrication of microfluidic systems areprovided in Manz et al., Trends in Anal. Chem. (1990) 10(5):144-149 andManz et al., Avd. in Chromatog. (1993) 33:1-66, which describe thefabrication of such fluidic devices and particularly microcapillarydevices, in silicon and glass substrates.

Applications of microfluidic systems are myriad. For example,International Patent Appln. WO 96/04547, published Feb. 15, 1996,describes the use of microfluidic systems for capillary electrophoresis,liquid chromatography, flow injection analysis, and chemical reactionand synthesis. U.S. patent application Ser. No. 08/671,987, filed Jun.28, 1996, and incorporated herein by reference, discloses wide rangingapplications of microfluidic systems in rapidly assaying large number ofcompounds for their effects on chemical, and preferably, biochemicalsystems. The phrase, "biochemical system," generally refers to achemical interaction which involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signaling and other reactions. Biochemicalsystems of particular interest include, e.g., receptor-ligandinteractions, enzyme-substrate interactions, cellular signalingpathways, transport reactions involving model barrier systems (e.g.,cells or membrane fractions) for bioavailability screening, and avariety of other general systems.

Many methods have been described for the transport and direction offluids, e.g., samples, analytes, buffers and reagents, within thesemicrofluidic systems or devices. One method moves fluids withinmicrofabricated devices by mechanical micropumps and valves within thedevice. See, Published U.K. Patent Application No. 2 248 891 (Oct. 18,1990), Published European Patent Application No. 568 902 (May 2, 1992),U.S. Pat. Nos. 5,271,724 (Aug. 21, 1991) and 5,277,556 (Jul. 3, 1991).See also, U.S. Pat. No. 5,171,132 (Dec. 21, 1990) to Miyazaki et al.Another method uses acoustic energy to move fluid samples within devicesby the effects of acoustic streaming. See, Published PCT Application No.94/05414 to Northrup and White. A straightforward method appliesexternal pressure to move fluids within the device. See, e.g., thediscussion in U.S. Pat. No. 5,304,487 to Wilding et al.

Still another method uses electric fields to move fluid materialsthrough the channels of the microfluidic system. See, e.g., PublishedEuropean Patent Application No. 376 611 (Dec. 30, 1988) to Kovacs,Harrison et al., Anal. Chem, (1992) 64:1926-1932 and Manz et al. J.Chromatog. (1992) 593:253-258, U.S. Pat. No. 5,126,022 to Soane.Electrokinetic forces have the advantages of direct control, fastresponse and simplicity. However, there are still some disadvantages.For maximum efficiency, it is desirable that the subject materials betransported as closely together as possible. Nonetheless, the materialsshould be transported without cross-contamination from other transportedmaterials. Further, the materials in one state at one location in amicrofluidic system should remain in the same state after being moved toanother location in the microfluidic system. These conditions permit thetesting, analysis and reaction of the compound materials to becontrolled, when and where as desired.

In a microfluidic system in which the materials are moved byelectrokinetic forces, the charged molecules and ions in the subjectmaterial regions and in the regions separating these subject materialregions are subjected to various electric fields to effect fluid flow.

Upon application of these electric fields, however; differently chargedspecies within the subject material will exhibit differentelectrophoretic mobilities, i.e., positively charged species will moveat a different rate than negatively charged species. In the past, theseparation of different species within a sample that was subjected to anelectric field was not considered a problem, but was, in fact, thedesired result, e.g., in capillary electrophoresis. However, wheresimple fluid transport is desired, these varied mobilities can result inan undesirable alteration or "electrophoretic bias" in the subjectmaterial.

Without consideration and measures to avoid cross-contamination, themicrofluidic system must either widely separate the subject materials,or, in the worst case, move the materials one at a time through thesystem. In either case, efficiency of the microfluidic system ismarkedly reduced. Furthermore, if the state of the transported materialscannot be maintained in transport, then many applications which requirethe materials to arrive at a location unchanged must be avoided.

The present invention solves or substantially mitigates these problemsof electrokinetic transport. With the present invention, microfluidicsystems can move materials efficiently and without undesired change inthe transported materials. The present invention presents a highthroughput microfluidic system having direct, fast and straightforwardcontrol over the movement of materials through the channels of themicrofluidic system with a wide range of applications, such as in thefields of chemistry, biochemistry, biotechnology, molecular biology andnumerous other fields.

SUMMARY OF THE INVENTION

The present invention provides for a microfluidic system whichelectroosmotically moves subject material along channels in fluid slugs,also termed "subject material regions," from a first point to a secondpoint in the microfluidic system. A first spacer region of high ionicconcentration contacts each subject material region on at least one sideand second spacer regions of low ionic concentration are arranged withthe subject material regions of subject material and first or high ionicconcentration spacer regions so that at least one low ionicconcentration region is always between the first and second points toensure that most of the voltage drop and resulting electric fieldbetween the two points is across the low ionic concentration region.

The present invention also provides for a electropipettor which iscompatible with a microfluidic system which moves subject materials withelectroosmotic forces. The electropipettor has a capillary having achannel. An electrode is attached along the outside length of thecapillary and terminates in a electrode ring at the end of thecapillary. By manipulating the voltages on the electrode and theelectrode at a target reservoir to which the channel is fluidlyconnected when the end of the capillary is placed into a materialsource, materials are electrokinetically introduced into the channel. Atrain of subject material regions, high and low ionic concentrationbuffer or spacer regions can be created in the channel for easyintroduction into the microfluidic system.

The present invention further compensates for electrophoretic bias asthe subject materials are electrokinetically transported along thechannels of a microfluidic system. In one embodiment a channel betweentwo points of the microfluidic system has two portions with sidewalls ofopposite surface charges. An electrode is placed between the twoportions. With the voltages at the two points substantially equal andthe middle electrode between the two portions set differently,electrophoretic forces are in opposite directions in the two portions,while electroosmotic forces are in the same direction. As subjectmaterial is transported from one point to the other, electrophoreticbias is compensated for, while electroosmotic forces move the fluidmaterials through the channel.

In another embodiment a chamber is formed at the intersection ofchannels of a microfluidic system. The chamber has sidewalls connectingthe sidewalls of the intersecting channels. When a subject materialregion is diverted from one channel into another channel at theintersection, the chamber sidewalls funnel the subject material regioninto the second channel. The width of the second channel is such thatdiffusion mixes any subject material which had been electrophoreticallybiased in the subject material region as it traveled along the firstchannel.

In still a further embodiment, the present invention provides amicrofluidic system and method of using that system for controllablydelivering a fluid stream within a microfluidic device having at leasttwo intersecting channels. The system includes a substrate having the atleast two intersecting channels disposed therein. In this aspect, theone of the channels is deeper than the other channel. The system alsoincludes an electroosmotic fluid direction system. The system isparticularly useful where the fluid stream comprises at least two fluidregions having different ionic strengths.

The present invention also provides a sampling system using theelectropipettor of the invention. The sampling system includes a samplesubstrate, which has a plurality of different samples immobilizedthereon. Also included is a translation system for moving theelectropipettor relative to said sample substrate.

The invention as hereinbefore described may be put into a plurality ofdifferent uses, which are themselves inventive, for example, as follows:

The use of a substrate having a channel, in transporting at least afirst subject material from at least a first location to a secondlocation along the channel, utilizing at least one region of low ionicconcentration which is transported along the channel due to an appliedvoltage.

A use of the aforementioned invention, in which the ionic concentrationof the one region is substantially lower than that of the subjectmaterial.

A use of the aforementioned invention, wherein a plurality of subjectmaterials are transported, separated by high ionic concentration spacerregions.

The use of a substrate having a channel along which at least a firstsubject material may be transported, in electrophoretic biascompensation, the channel being divided into a first and a secondportion, in which the wall or walls of the channel are oppositelycharged, such that electrophoretic bias on the at least first subjectmaterial due to transportation in the first portion is substantiallycompensated for by electrophoretic bias due to transport in the secondportion.

A use of the aforementioned invention in which a first electrode islocated at a remote end of the first portion, a second electrode islocated at the intersection between the portions and a third electrodeis located at a remote end of the second portion.

A use of the aforementioned invention, in which the substrate is amicrofluidic system.

A use of the aforementioned invention in which the substrate is anelectropipettor.

A use of the aforementioned invention, in which the electropipettor hasa main channel for transportation of the subject material and at leastone further channel fluidly connected to the main channel from which afurther material to be transported along the main channel is obtained.

A use of the aforementioned invention, in which the further material isdrawn into the main channel as a buffer region between each of aplurality of separate subject materials.

The use of a microfluidic system having at least a first and a secondfluid channel which intersect, in optimizing flow conditions, thechannels having different depths.

A use of the aforementioned invention in which one channel is between 2to 10 times deeper than the other channel.

The use of a microfluidic system having a first channel and a secondchannel intersecting the first channel, in electrophoretic compensation,the intersection between the channels being shaped such that a fluidbeing transported along the first channel towards the second channel ismixed at the intersection and any electrophoretic bias in the fluid isdissipated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic illustration of one embodiment of amicrofluidic system;

FIG. 2A illustrates an arrangement of fluid regions traveling in achannel of the microfluidic system of FIG. 1, according to oneembodiment of the present invention; FIG. B is a scaled drawing ofanother arrangement of different fluid regions traveling in a channel ofthe microfluidic system according to the present invention;

FIG. 3A is another arrangement with high ionic concentration spacerregions before a subject material region traveling within a channel ofthe microfluidic system; FIG. 3B shows an arrangement with high ionicconcentration spacer regions after a subject material region travelingin a channel of the microfluidic system;

FIG. 4A is a schematic diagram of one embodiment of a electropipettoraccording to the present invention; FIG. 4B is a schematic diagram ofanother electropipettor according to the present invention;

FIG. 5 is a schematic diagram of a channel having portions withoppositely charged sidewalls in a microfluidic system, according to thepresent invention; and

FIGS. 6A-6D illustrate the mixing action of funneling sidewalls at theintersection of channels in a microfluidic system, according to thepresent invention.

FIG. 7A shows the results of three injections of a sample fluid made upof two oppositely charged chemical species in a low salt buffer, into acapillary filled with low salt buffer. FIG. 7B shows the results ofthree sample injections where the sample is in high salt buffer, highsalt buffer fluids were injected at either end of the sample region tofunction as guard bands, and the sample/guard bands were run in a lowsalt buffer filled capillary. FIG. 7C shows the results of three sampleinjections similar to that of FIG. 7B except that the size of the lowsalt spacer region between the sample/high salt spacers (guard bands) isreduced, allowing partial resolution of the species within the sample,without allowing the sample elements to compromise subsequent orprevious samples.

FIG. 8 shows a schematic illustration of an electropipettor for use witha sampling system using samples immobilized, e.g., dried, on a substratesheet or matrix.

FIG. 9A is a plot of fluorescence versus time which illustrates themovement of a sample fluid made up of test chemical species which isperiodically injected into, and moved through, an electropipettor,according to the present invention. FIG. 9B is another plot which showsthe movement of the sample fluid with the chemical species through amicrofluidic substrate which is connected to the electropipettor, underdifferent parameters. FIG. 9C is a plot which illustrates the movementof the sample fluid and chemical species through an electropipettorformed from an air abraded substrate.

FIG. 10 is a plot which again illustrates the movement of a chemicalspecies in a sample fluid which has been periodically injected into anelectropipettor, according to the present invention. In this experiment,the species is a small molecule compound.

DETAILED DESCRIPTION OF THE INVENTION

I. General Organization of a Microfluidic System

FIG. 1 discloses a representative diagram of an exemplary microfluidicsystem 100 according to the present invention. As shown, the overalldevice 100 is fabricated in a planar substrate 102. Suitable substratematerials are generally selected based upon their compatibility with theconditions present in the particular operation to be performed by thedevice. Such conditions can include extremes of pH, temperature, ionicconcentration, and application of electrical fields. Additionally,substrate materials are also selected for their inertness to criticalcomponents of an analysis or synthesis to be carried out by the system.

Useful substrate materials include, e.g., glass, quartz and silicon, aswell as polymeric substrates, e.g., plastics. In the case of conductiveor semiconductive substrates, there should be an insulating layer on thesubstrate. This is particularly important where the device incorporateselectrical elements, e.g., electrical fluid direction systems, sensorsand the like, or uses electroosmotic forces to move materials about thesystem, as discussed below. In the case of polymeric substrates, thesubstrate materials may be rigid, semi-rigid, or non-rigid, opaque,semi-opaque or transparent, depending upon the use for which they areintended. For example, devices which include an optical or visualdetection element, will generally be fabricated, at least in part, fromtransparent materials to allow, or at least, facilitate that detection.Alternatively, transparent windows of, e.g., glass or quartz, may beincorporated into the device for these types of detection elements.Additionally, the polymeric materials may have linear or branchedbackbones, and may be crosslinked or non-crosslinked. Examples ofparticularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC),polystyrene, polysulfone, polycarbonate and the like.

The system shown in FIG. 1 includes a series of channels 110, 112, 114and 116 fabricated into the surface of the substrate 102. As discussedin the definition of "microfluidic," these channels typically have verysmall cross sectional dimensions, preferably in the range of from about0.1 μm to about 100 μm. For the particular applications discussed below,channels with depths of about 10 μm and widths of about 60 μm workeffectively, though deviations from these dimensions are also possible.

Manufacturing of these channels and other microscale elements into thesurface of the substrate 102 may be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques may be employed in fabricating glass, quartz orsilicon substrates, for example, with methods well known in thesemiconductor manufacturing industries. Photolithographic masking,plasma or wet etching and other semiconductor processing technologiesdefine microscale elements in and on substrate surfaces. Alternatively,micromachining methods, such as laser drilling, micromilling and thelike, may be employed. Similarly, for polymeric substrates, well knownmanufacturing techniques may also be used. These techniques includeinjection molding techniques or stamp molding methods where largenumbers of substrates may be produced using, e.g., rolling stamps toproduce large sheets of microscale substrates, or polymer microcastingtechniques where the substrate is polymerized within a microfabricatedmold.

Besides the substrate 102, the microfluidic system includes anadditional planar element (not shown) which overlays the channeledsubstrate 102 to enclose and fluidly seal the various channels to formconduits. The planar cover element may be attached to the substrate by avariety of means, including, e.g., thermal bonding, adhesives or, in thecase of glass, or semi-rigid and non-rigid polymeric substrates, anatural adhesion between the two components. The planar cover elementmay additionally be provided with access ports and/or reservoirs forintroducing the various fluid elements needed for a particular screen.

The system 100 shown in FIG. 1 also includes reservoirs 104, 106 and108, which are disposed and fluidly connected at the ends of thechannels 114, 116 and 110 respectively. As shown, sample channel 112, isused to introduce a plurality of different subject materials into thedevice. As such, the channel 112 is fluidly connected to a source oflarge numbers of separate subject materials which are individuallyintroduced into the sample channel 112 and subsequently into anotherchannel 110. As shown, the channel 110 is used for analyzing the subjectmaterials by electrophoresis. It should be noted that the term, "subjectmaterials," simply refers to the material, such as a chemical orbiological compound, of interest. Subject compounds may include a widevariety of different compounds, including chemical compounds, mixturesof chemical compounds, e.g., polysaccharides, small organic or inorganicmolecules, biological macromolecules, e.g., peptides, proteins, nucleicacids, or extracts made from biological materials, such as bacteria,plants, fungi, or animal cells or tissues, naturally occurring orsynthetic compositions.

The system 100 moves materials through the channels 110, 112, 114 and116 by electrokinetic forces which are provided by a voltage controllerthat is capable of applying selectable voltage levels, simultaneously,to each of the reservoirs, including ground. Such a voltage controllercan be implemented using multiple voltage dividers and multiple relaysto obtain the selectable voltage levels. Alternatively, multipleindependent voltage sources may be used. The voltage controller iselectrically connected to each of the reservoirs via an electrodepositioned or fabricated within each of the plurality of reservoirs.See, for example, published International Patent Application No. WO96/04547 to Ramsey, which is incorporated herein by reference in itsentirety for all purposes.

II. Electrokinetic Transport

A. Generally

The electrokinetic forces on the fluid materials in the channels of thesystem 100 may be separated into electroosmotic forces andelectrophoretic forces. The fluid control systems used in the system ofthe present invention employ electroosmotic force to move, direct andmix fluids in the various channels and reaction chambers present on thesurface of the substrate 102. In brief, when an appropriate fluid isplaced in a channel or other fluid conduit having functional groupspresent at the surface, those groups can ionize. Where the surface ofthe channel includes hydroxyl functional groups at the surface, forexample, protons can leave the surface of the channel and enter thefluid. Under such conditions, the surface possesses a net negativecharge, whereas the fluid possesses an excess of protons or positivecharge, particularly localized near the interface between the channelsurface and the fluid.

By applying an electric field across the length of the channel, cationsflow toward the negative electrode. Movement of the positively chargedspecies in the fluid pulls the solvent with them. The steady statevelocity of this fluid movement is generally given by the equation:##EQU1## where v is the solvent velocity, .di-elect cons. is thedielectric constant of the fluid, .di-elect cons. is the zeta potentialof the surface, Ε is the electric field strength, and η is the solventviscosity. Thus, as can be easily seen from this equation, the solventvelocity is directly proportional to the zeta potential and the appliedfield.

Besides electroosmotic forces, there are also electrophoretic forceswhich affect charged molecules as they move through the channels of thesystem 100. In the transport of subject materials from one point toanother point in the system 100, it is often desirable for thecomposition of the subject materials to remain unaffected in thetransport, i.e., that the subject materials are not electrophoreticallydifferentiated in the transport.

In accordance with the present invention, the subject materials aremoved about the channels as slugs of fluid (hereafter termed "subjectmaterial regions"), which have a high ionic concentration to minimizeelectrophoretic forces on the subject materials within these particularregions. To minimize the effect of electrophoretic forces within thesubject material regions, regions of spacer fluids ("first spacerregions") are placed on either side of a slug. These first spacerregions have a high ionic concentration to minimize the electric fieldsin these regions, as explained below, so that subject materials areessentially unaffected by the transport from one location to anotherlocation in a microfluidic system. The subject materials are transportedthrough the representative channels 110, 112, 114, 116 of the system 100in regions of certain ionic strengths, together with other regions ofionic strengths varying from those regions bearing the subjectmaterials.

A specific arrangement is illustrated in FIG. 2A, which illustratessubject material regions 200 being transported from point A to point Balong a channel of the microfluidic system 100. In either side of thesubject material regions 200 are first spacer regions 201 of high ionicstrength fluid. Additionally, second spacer regions 202 of low ionicconcentration fluid periodically separate arrangements of subjectmaterial regions 200 and first spacer regions 201. Being of low ionicconcentration, most of the voltage drop between points A and B occursacross these second spacer regions 202. The second or low concentrationspacer regions 202 are interspersed between the arrangements of subjectmaterial region 200 and first spacer region 201 such that, as thesubject material regions 200 and the first spacer regions 201 areelectroosmotically pumped through the channel, there is always at leastone second or low ionic concentration spacer region 202 between thepoints A and B. This ensures that most of the voltage drop occurs in thesecond spacer region 202, rather than across the subject material region200 and first spacer regions 201. Stated differently, the electric fieldbetween points A and B is concentrated in the second spacer region 202and the subject material regions 200 and first spacer regions 201experience low electric fields (and low electrophoretic forces). Thus,depending upon the relative ionic concentrations in the subject materialregions 200, first spacer regions 201 and second or low ionicconcentration spacer regions 202, other arrangements of these subjectmaterial regions 200, and first and second spacer regions 201 and 202can be made.

For example, FIG. 2B illustrates an arrangement in which a second or lowionic concentration spacer region 202 is regularly spaced between eachcombination of first spacer region 201/subject material region 200/firstspacer region 201. Such an arrangement ensures that there is always atleast one second or low concentration spacer region 202 between points Aand B. Furthermore, the drawings are drawn to scale to illustrate therelative lengths of a possible combination of subject material region200, first or high concentration spacer region 201 and second or lowconcentration spacer region 202. In the example of FIG. 2B, the subjectmaterial region 200 holds the subject material in a high ionicconcentration of 150 mM of NaCl. The subject material region 200 is 1 mmlong in the channel. The two first spacer regions 201 have ionicconcentrations of 150 mM of NaCl. Each first spacer region 201 is 1 mmlong. The second spacer region 202 is 2 mm and has an ionicconcentration of 5 mM of borate buffer. This particular configuration isdesigned to maintain a rapidly electrophoresing compound in the subjectmaterial region 200 and buffer regions 201 while the compound travelsthrough the channels of the microfluidic system. For example, usingthese methods, a subject material region containing, e.g., benzoic acid,can be flowed through a microfluidic system for upwards of 72 secondswithout suffering excessive electrophoretic bias.

Stated more generally, the velocity of fluid flow, V_(EoF), through thechannels of the microfluidic system can be determined and, bymeasurement, it is possible to determine the total distance, l_(T),which a subject matter molecule is to travel through the channels. Thusthe transit time, t_(Tr), for the subject matter molecule to travel thetotal distance is:

    t.sub.Tr =l.sub.T /V.sub.EoF

To contain a subject matter molecule x within the first spacer region201 next to the subject material region 200, the length of the firstspacer region 201, l_(g), should be greater than the electrophoreticvelocity of the subject matter molecule x in the first spacer region201, V_(gx), multiplied by the transit time:

    l.sub.g >(V.sub.gx)(t.sub.Tr)

Since the electrophoretic velocity is proportional to the electric fieldin the first spacer region 201, the present invention allows controlover V_(gx) so that the subject materials can be contained in transportthrough the microfluidic system channels.

In the arrangements in FIGS. 2A and 2B, the first or high ionicconcentration spacer regions 201 help maintain the position of thesubject materials in the vicinity of its subject material region 200. Nomatter what the polarity of the charges of the subject material, thefirst spacer regions 201 on either side of the subject material region200 ensures that any subject material leaving the subject materialregion 200 is only subject to a low electric field due to the relativehigh ionic concentrations in the first spacer regions 201. If thepolarity of the subject material is known, then the direction of theelectrophoretic force on the molecules of the subject material is alsoknown.

FIG. 3A illustrates an example where charges of the subject material inall the subject material regions 200 are such that the electrophoreticforce on the subject material molecules are in the same direction as thedirection of the electroosmotic flow. Hence the first spacer regions 201precede the subject material regions 200 in the direction of flow. Thereare no first spacer regions 201 following the subject material regions200 because the electrophoretic force keeps the subject material fromescaping the subject material region 200 in that direction. Byeliminating one-half of the first spacer regions 201, more subjectmaterial regions 200 with their subject material can be carried perchannel length. This enhances the transportation efficiency of themicrofluidic system. The second or low ionic concentration spacerregions 202 are arranged with respect to the subject material regions200 and the first or high ionic concentration spacer regions 201 so thathigh electric fields fall in the second spacer regions 202 and theelectric fields (and electrophoretic forces) in the subject materialregions 200 and first spacer regions 201 are kept low.

In FIG. 3B the first spacer regions 201 follow the subject materialregions 200 in the direction of the electroosmotic flow. In thisexample, the charges of the subject material in all the subject materialregions 200 are such that the electrophoretic force on the subjectmatter molecules are in the opposite direction as the direction of theelectroosmotic flow. Hence the subject material may escape the confinesof its subject material region, in effect, being left behind by itssubject material region 200. The first spacer regions 201 following thesubject material regions 200 keep the subject material from migratingtoo far from its subject material region 200. Likewise, the second orlow ionic concentration spacer regions 202 are arranged with the subjectmaterial regions 200 and the first or high ionic concentration spacerregions 201 so that high electric fields fall in the second spacerregions 202 and the electric fields in the subject material regions 200and first spacer regions 201 are kept low.

Various high and low ionic strength solutions are selected to produce asolution having a desired electrical conductivity for the first andsecond spacer regions 201 and 202. The specific ions that impartelectrical conductivity to the solution maybe derived from inorganicsalts (such as NaCl, KI, CaCl₂, FeF₃, (NH₄)₂ SO₄ and so forth), organicsalts (such as pyridinium benzoate, benzalkonium laurate), or mixedinorganic/organic salts (such as sodium benzoate, sodium deoxylsulfate,benzylaminehydrochloride). These ions are also selected to be compatiblewith the chemical reactions, separations, etc. to be carried out in themicrofluidic system. In addition to aqueous solvents, mixtures ofaqueous/organic solvents, such as low concentrations of DMSO in water,may be used to assist in the solubilization of the subject mattermolecules. Mixtures of organic solvents, such as CHCl₃ :MeOH, may bealso used for the purpose of accelerating assays for phospholipaseactivity, for example.

Generally, when aqueous solvents are used, solution conductivity isadjusted using inorganic ions. When less polar solvents are used,organic or mixed inorganic/organic ions are typically used. In caseswhere two immiscible solvents may be simultaneously present (e.g., waterand a hydrocarbon such as decane) such that electrical current must flowfrom one into the other, ionophores (e.g., valinomycin, nonactin,various crown ethers, etc.) and their appropriate ions may be used toconduct current through the non-polar solvent.

B. Electrokinetic Control of Pressure Based Flow

In the electrokinetic flow systems described herein, the presence ofdifferentially mobile fluids (e.g., having a different electrokineticmobility in the particular system) in a channel may result in multipledifferent pressures being present along the length of a channel in thesystem. For example, these electrokinetic flow systems typically employa series of regions of low and high ionic concentration fluids (e.g.,first and second spacer regions and subject material regions of subjectmaterial) in a given channel to effect electroosmotic flow, while at thesame time, preventing effects of electrophoretic bias within a subjectmaterial containing subject material region. As the low ionicconcentration regions within the channel tend to drop the most appliedvoltage across their length, they will tend to push the fluids through achannel. Conversely, high ionic concentration fluid regions within thechannel provide relatively little voltage drop across their lengths, andtend to slow down fluid flow due to viscous drag.

As a result of these pushing and dragging effects, pressure variationscan generally be produced along the length of a fluid filled channel.The highest pressure is typically found at the front or leading edge ofthe low ionic concentration regions (e.g., the second spacer regions),while the lowest pressure is typically found at the trailing or backedge of these low ionic strength fluid regions.

While these pressure differentials are largely irrelevant in straightchannel systems, their effects can result in reduced control over fluiddirection and manipulation in microfluidic devices that employintersecting channel arrangements i.e., the systems described in U.S.patent application Ser. No. 08/671,987, previously incorporated byreference. For example, where a second channel is configured tointersect a first channel which contains fluid regions of varying ionicstrength, the pressure fluctuations described above can cause fluid toflow in and out of the intersecting second channel as these differentfluid regions move past the intersection. This fluctuating flow couldpotentially, significantly disturb the quantitative electroosmoticallydriven flow of fluids from the second channel, and/or perturb thevarious fluid regions within the channel.

By reducing the depth of the intersecting channel, e.g., the secondchannel, relative to the first or main channel, the fluctuations influid flow can be substantially eliminated. In particular, inelectroosmotic fluid propulsion or direction, for a given voltagegradient, the rate of flow (volume/time) generally varies as thereciprocal of the depth of the channel for channels having an aspectratio of >10 (width:depth). With some minor, inconsequential error forthe calculation, this general ratio also holds true for lower aspectratios, e.g., aspect ratios >5. Conversely, the pressure induced flowfor the same channel will vary as the third power of the reciprocal ofthe channel depth. Thus, the pressure build-up in a channel due to thesimultaneous presence of fluid regions of differing ionic strength willvary as the square of the reciprocal of the channel depth.

Accordingly, by decreasing the depth of the intersecting second channelrelative to the depth of the first or main channel by a factor of X, onecan significantly reduce the pressure induced flow, e.g., by a factor ofX³, while only slightly reducing the electroosmotically induced flow,e.g., by a factor of X. For example, where the second channel is reducedin depth relative to the first channel by one order of magnitude, thepressure induced flow will be reduced 1000 times while theelectroosmotically induced flow will be reduced by only a factor of ten.Accordingly, in some aspects, the present invention providesmicrofluidic devices as generally described herein, e.g., having atleast first and second intersecting channels disposed therein, but wherethe first channel is deeper than the second channel. Generally, thedepths of the channels may be varied to obtain optimal flow conditionsfor a desired application. As such, depending upon the application, thefirst channel may be greater than about two times as deep as the secondchannel, greater than about 5 times as deep as the second channel, andeven greater than about ten times as deep as the second channel.

In addition to their use in mitigating pressure effects, varied channeldepths may also be used to differentially flow fluids within differentchannels of the same device, e.g., to mix different proportions offluids from different sources, and the like.

III. Electropipettor

As described above, any subject material can be transported efficientlythrough the microfluidic system 100 in or near the subject materialregions 200. With the first and second spacer regions 201 and 202, thesubject materials are localized as they travel through the channels ofthe system. For efficient introduction of subject matter into amicrofluidic system, the present invention also provides anelectropipettor which introduces subject material into a microfluidicsystem in the same serial stream of combinations of subject materialregion 200, first and second spacer regions 201 and 202.

A. Structure and Operation

As illustrated in FIG. 4A, an electropipettor 250 is formed by a hollowcapillary tube 251. The capillary tube 251 has a channel 254 with thedimensions of the channels of the microfluidic system 100 to which thechannel 254 is fluidly connected. As shown in FIG. 4A, the channel 254is a cylinder having a cross-sectional diameter in the range of 1-100μm, with a diameter of approximately 30 μm being preferable. Anelectrode 252 runs down the outside wall of the capillary tube 251 andterminates in a ring electrode 253 around the end of the tube 251. Todraw the subject materials in the subject material regions 200 with thebuffer regions 201 and 202 into the electropipettor channel 254, theelectrode 252 is driven to a voltage with respect to the voltage of atarget reservoir (not shown) which is fluidly connected to the channel254. The target reservoir is in the microfluidic system 100 so that thesubject material regions 200 and the buffer regions 201 and 202 alreadyin the channel 254 are transported serially from the electropipettorinto the system 100.

Procedurally, the capillary channel end of the electropipettor 250 isplaced into a source of subject material. A voltage is applied to theelectrode 252 with respect to an electrode in the target reservoir. Thering electrode 253, being placed in contact with the subject materialsource, electrically biases the source to create a voltage drop betweenthe subject material source and the target reservoir. In effect, thesubject material source and the target reservoir become Point A and B ina microfluidic system, i.e., as shown in FIG. 2A. The subject materialis electrokinetically introduced into the capillary channel 254 tocreate a subject material region 200. The voltage on the electrode 252is then turned off and the capillary channel end is placed into a sourceof buffer material of high ionic concentration. A voltage is againapplied to the electrode 252 with respect to the target reservoirelectrode such that the first spacer region 201 is electrokineticallyintroduced into the capillary channel 254 next to the subject materialregion 200. If a second or low ionic concentration spacer region 202 isthen desirable in the electropipettor channel 254, the end of thecapillary channel 254 is inserted into a source of low ionicconcentration buffer material and a voltage applied to the electrode252. The electropipettor 250 can then move to another source of subjectmaterial to create another subject material region 200 in the channel254.

By repeating the steps above, a plurality of subject material regions200 with different subject materials, which are separated by first andsecond spacer regions 201 and 202, are electrokinetically introducedinto the capillary channel 254 and into the microfluidic system 100.

Note that if the sources of the subject material and the buffermaterials (of low and high ionic concentration) have their ownelectrode, the electrode 252 is not required. Voltages between thetarget reservoir and the source electrodes operate the electropipettor.Alternatively, the electrode 252 might be in fixed relationship with,but separated from, the capillary tube 251 so that when the end of thetube 251 contacts a reservoir, the electrode 252 also contacts thereservoir. Operation is the same as that described for the FIG. 4Aelectropipettor.

FIG. 4B illustrates a variation of the electropipettor 250 of FIG. 4A.In this variation the electropipettor 270 is not required to travelbetween a subject material source and buffer material sources to createthe first and second spacer regions 201 and 202 within the pipettor. Theelectropipettor 270 has a body 271 with three capillary channels 274,275 and 276. The main channel 274 operates identically to the channel254 of the previously described electropipettor 250. However, the twoauxiliary capillary channels 275 and 276 at one end are fluidlyconnected to buffer source reservoirs (not shown) and the other end ofthe channels 275 and 276 is fluidly connected to the main channel 274.One reservoir (i.e., connected to auxiliary channel 275) holds buffermaterial of high ionic concentration, and the other reservoir (i.e.,connected to the channel 276) holds buffer material of low ionicconcentration.

All of the reservoirs are connected to electrodes for electricallybiasing these reservoirs for operation of the electropipettor 270. Theelectropipettor 270 may also have an electrode 272 along the walls ofits body 271 which terminates in a ring electrode 273 at the end of themain channel 274. By applying voltages on the electrode 272 (and ringelectrode 273) to create voltage drops along the channels 274, 275, 276,not only can subject material be drawn into the main channel 274 fromsubject material sources, but buffer material of high and low ionicconcentrations can also be drawn from the auxiliary channels 275 and 276into the main channel 274.

To operate the electropipettor 270 with the electrode 272, the end ofthe main capillary channel 274 is placed into a source 280 of subjectmaterial. A voltage is applied to the electrode 272 with respect to anelectrode in the target reservoir to create a voltage drop between thesubject material source 280 and the target reservoir. The subjectmaterial is electrokinetically drawn into the capillary channel 274. Thecapillary channel end is then removed from the subject material source280 and a voltage drop is created between the target reservoir connectedto the channel 274 and the reservoir connected to the channel 275. Afirst or high ionic strength spacer region 201 is formed in the channel274. Capillary action inhibits the introduction of air into the channel274 as the buffer material is drawn from the auxiliary channel 275. If asecond or low ionic concentration spacer region 202 is then desired inthe main channel 274, a voltage is applied to the electrodes in thetarget reservoir and in the reservoir of low ionic concentration buffermaterial. A second spacer region 202 is electrokinetically introducedinto the capillary channel 274 from the second auxiliary channel 276 .The electropipettor 270 can then move to another source of subjectmaterial to create another subject material region 200 in the channel274.

By repeating the steps above, a plurality of subject material regions200 with different subject materials, which are separated by first andsecond spacer regions 201 and 202, are electrokinetically introducedinto the capillary channel 274 and into the microfluidic system 100.

If it is undesirable to expose the subject material source tooxidation/reduction reactions from the ring electrode 273, theelectropipettor may be operated without the electrode 272. Becauseelectroosmotic flow is slower in solutions of higher ionic strength, theapplication of a potential (- to +) from the reservoir connecting thechannel 274 to the reservoir connecting the channel 275 results in theformation of a vacuum at the point where the channels 274 and 275intersect. This vacuum draws samples from the subject material sourceinto the channel 274. When operated in this mode, the subject materialis somewhat diluted with the solutions in the channels 275 and 276 .This dilution can be mitigated by reducing the relative dimensions ofthe channels 276 and 275 with respect to the channel 274.

To introduce first and second spacer regions 201 and 202 into thecapillary channel 274, the electropipettor 270 is operated as describedabove. The capillary channel end is removed from the subject materialsource 280 and a voltage drop is created between the target reservoirfor the channel 274 and the reservoir connected to the selected channels275 or 276.

Although generally described in terms of having two auxiliary channelsand a main channel, it will be appreciated that additional auxiliarychannels may also be provided to introduce additional fluids, buffers,diluents, reagents and the like, into the main channel.

As described above for intersecting channels within a microfluidicdevice, e.g., chip, pressure differentials resulting from differentiallymobile fluids within the different pipettor channels also can affect thecontrol of fluid flow within the pipettor channel. Accordingly, asdescribed above, the various pipettor channels may also be providedhaving varied channel depths relative to each other, in order tooptimize fluid control.

B. Method of Electropipettor Manufacture

The electropipettor might be created from a hollow capillary tube, suchas described with respect to FIG. 4A. For more complex structures,however, the electropipettor is optimally formed from the same substratematerial as that of the microchannel system discussed above. Theelectropipettor channels (and reservoirs) are formed in the substrate inthe same manner as the microchannels for a microfluidic system and thechanneled substrate is covered by a planar cover element, also describedabove. The edges of the substrate and cover element may then be shapedto the proper horizontal dimensions of the pipettor, especially its end,as required. Techniques, such as etching, air abrasion (blasting asurface with particles and forced air), grinding and cutting, may beused. Electrodes are then created on the surface of the substrate andpossibly cover, as required. Alternatively, the edges of the substrateand the cover element may be shaped prior to being attached together.This method of manufacture is particularly suited for multichannelelectropipettors, such as described immediately above with respect toFIG. 4B and described below with respect to FIG. 8.

IV. Sampling System

As described above, the methods, systems and apparatuses described abovewill generally find widespread applicability in a variety ofdisciplines. For example, as noted previously, these methods and systemsmay be particularly well suited to the task of high throughput chemicalscreening in, e.g., drug discovery applications, such as is described incopending U.S. patent application Ser. No. 08/671,987, filed Jun. 28,1996, and previously incorporated by reference.

A. Sample Matrices

The pipetting and fluid transport systems of the invention are generallydescribed in terms of sampling numbers of liquid samples, i.e., frommulti-well plates. In many instances, however, the number or nature ofthe liquid based samples to be sampled may generate sample handlingproblems. For example, in chemical screening or drug discoveryapplications, libraries of compounds for screening may number in thethousands or even the hundreds of thousands. As a result, such librarieswould require extremely large numbers of sample plates, which, even withthe aid of robotic systems, would create myriad difficulties in samplestorage, manipulation and identification. Further, in some cases,specific sample compounds may degrade, complex or otherwise possessrelatively short active half-lives when stored in liquid form. This canpotentially result in suspect results where samples are stored in liquidform for long periods prior to screening.

Accordingly, the present invention provides sampling systems whichaddress these further problems, by providing the compounds to be sampledin an immobilized format. By "immobilized format" is meant that thesample material is provided in a fixed position, either by incorporationwithin a fixed matrix, i.e., porous matrix, charged matrix, hydrophobicor hydrophilic matrix, which maintains the sample in a given location.Alternatively, such immobilized samples include samples spotted anddried upon a given sample matrix. In preferred aspects, the compounds tobe screened are provided on a sample matrix in dried form. Typically,such sample matrices will include any of a number of materials that canbe used in the spotting or immobilization of materials, including, e.g.,membranes, such as cellulose, nitrocellulose, PVDF, nylon, polysulfoneand the like. Typically, flexible sample matrices are preferred, topermit folding or rolling of the sample matrices which have largenumbers of different sample compounds immobilized thereon, for easystorage and handling.

Generally, samples may be applied to the sample matrix by any of anumber of well known methods. For example, sample libraries may bespotted on sheets of a sample matrix using robotic pipetting systemswhich allow for spotting of large numbers of compounds. Alternatively,the sample matrix may be treated to provide predefined areas for samplelocalization, e.g., indented wells, or hydrophilic regions surrounded byhydrophobic barriers, or hydrophobic regions surrounded by hydrophilicbarriers (e.g., where samples are originally in a hydrophobic solution),where spotted materials will be retained during the drying process. Suchtreatments then allow the use of more advanced sample applicationmethods, such as those described in U.S. Pat. No. 5,474,796, wherein apiezoelectric pump and nozzle system is used to direct liquid samples toa surface. Generally, however, the methods described in the '796 patentare concerned with the application of liquid samples on a surface forsubsequent reaction with additional liquid samples. However, thesemethods could be readily modified to provide dry spotted samples on asubstrate.

Other immobilization or spotting methods may be similarly employed. Forexample, where samples are stable in liquid form, sample matrices mayinclude a porous layer, gel or other polymer material which retain aliquid sample without allowing excess diffusion, evaporation or thelike, but permit withdrawal of at least a portion of the samplematerial, as desired. In order to draw a sample into the pipettor, thepipettor will free a portion of the sample from the matrix, e.g., bydissolving the matrix, ion exchange, dilution of the sample, and thelike.

B. Resolubilizing Pipettor

As noted, the sampling and fluid transport methods and systems of thepresent invention are readily applicable to screening, assaying orotherwise processing samples immobilized in these sample formats. Forexample, where sample materials are provided in a dried form on a samplematrix, the electropipetting system may be applied to the surface of thematrix. The electropipettor is then operated to expel a small volume ofliquid which solubilizes the previously dried sample on the matrixsurface (dissolves a retaining matrix, or elutes a sample from animmobilizing support), e.g., by reversing the polarity of the fieldapplied to the pipettor, or by applying a potential from the low ionicconcentration buffer reservoir to the high ionic concentration bufferreservoir, as described above. Once the sample is resolubilized, thepipettor is then operated in its typical forward format to draw thesolubilized sample into the pipettor channel as previously described.

A schematic illustration of one embodiment of an electropipettor usefulin performing this function, and its operation, is shown in FIG. 8.Briefly, the top end 802 of the pipettor (as shown) 800 is generallyconnected to the assay system, e.g., a microfluidic chip, such thatvoltages can be supplied independently to the three channels of thepipettor 804, 806 and 808. Channels 804 and 808 are typically fluidlyconnected to buffer reservoirs containing low and high ionicconcentration fluids, respectively. In operation, the tip of thepipettor 810 is contacted to the surface of a sample matrix 812 where animmobilized(e.g., dried) sample 814 is located. A voltage is appliedfrom the low ionic concentration buffer channel 804 to the high ionicconcentration buffer channel 808, such that buffer is forced out of theend of the pipettor tip to contact and dissolve the sample. As shown,the pipettor tip 816 may include a recessed region or "sample cup" 818in order to maintain the expelled solution between the pipettor tip andthe matrix surface. In some cases, e.g., where organic samples are beingscreened, in order to ensure dissolution of the sample, an appropriateconcentration of an acceptable solvent, e.g., DMSO, may be included withthe low ionic concentration buffer. Voltage is then applied from thehigh ionic concentration buffer channel to the sample channel 806 todraw the sample into the pipettor in the form of a sample plug 820. Oncethe sample is completely withdrawn from the sample cup into thepipettor, the high surface tension resulting from air entering thesample channel will terminate aspiration of the sample, and high ionicconcentration buffer solution will begin flowing into the sample channelto form a first spacer region 822, following the sample. Low ionicconcentration buffer solution may then be injected into the samplechannel, i.e., as a second spacer region 824, by applying the voltagefrom the low ionic concentration buffer channel 804 to the samplechannel 806. Prior to or during presentation of the next sample positionon the matrix, a first or high ionic concentration spacer region 822 maybe introduced into the sample channel by applying the voltage betweenthe high ionic concentration buffer channel and the sample channel. Asnoted previously, a roll, sheet, plate, or numerous rolls, sheets orplates, of sample matrix having thousands or hundreds of thousands ofdifferent compounds to be screened, may be presented in this manner,allowing their serial screening in an appropriate apparatus or system.

V. Elimination of Electrophoretic Bias

As explained above, electrokinetic forces are used to transport thesubject material through the channels of the microfluidic system 100. Ifthe subject material is charged in solution, then it is subject to notonly electroosmotic forces, but also to electrophoretic forces. Thus thesubject material is likely to have undergone electrophoresis intraveling from one point to another point along a channel of themicrofluidic system. Hence the mixture of the subject material or thelocalization of differently charged species in a subject material region200 at the starting point is likely to be different than the mixture orlocalization at the arrival point. Furthermore, there is the possibilitythat the subject material might not even be in the subject materialregion 200 at the arrival point, despite the first spacer regions 201.

Therefore, another aspect of the present invention compensates forelectrophoretic bias as the subject materials are transported throughthe microfluidic system 100. One way to compensate for electrophoreticbias is illustrated in FIG. 5. In the microfluidic system 100 describedabove, each of the channels 110, 112, 114 and 116 was considered as aunitary structure along its length. In FIG. 5, an exemplary channel 140is divided into two portions 142 and 144. The sidewalls of each channelportion 142 and 144 have surface charges which are of opposite polarity.The two channel portions 142 and 144 are physically connected togetherby a salt bridge 133, such as a glass frit or gel layer. While the saltbridge 133 separates the fluids in the channel 140 from an ionic fluidin a reservoir 135 which is partially defined by the salt bridge 133,the salt bridge 133 permits ions to pass through. Hence the reservoir135 is in electrical, but not fluid, communication with the channel 140.

To impart electroosmotic and electrophoretic forces along the channel140 between points A and B, electrodes 132 and 134 are arranged atpoints A and B respectively. Additionally, a third electrode 137 isplaced in the reservoir 135 at the junction of the two portions 142 and144. The electrodes 132 and 134 are maintained at the same voltage andthe electrode 137 at another voltage. In the example illustrated in FIG.5, the two electrodes 132 and 134 are at a negative voltage, while theelectrode 137 and hence the junction of the two portions 142 and 144 areat zero voltage, i.e., ground. Thus the voltage drops, and hence theelectric fields, in the portions 142 and 144 are directed in oppositedirections. Specifically, the electric fields point away from eachother. Thus the electrophoretic force on a particularly charged moleculeis in one direction in the channel portion 142 and in the oppositedirection in the channel portion 144. Any electrophoretic bias on asubject material is compensated for after traveling through the twoportions 142 and 144.

The electroosmotic force in both portions 142 and 144 are still in thesame direction, however. For example, assuming that the sidewalls of thechannel portion 142 have positive surface charges, which attractnegative ions in solution, and the sidewalls of the channel portion 144have negative surface charges, which attract positive ions in solution,as shown in FIG. 5, the electroosmotic force in both portions 142 and144 is to the right of the drawing. Thus the subject material istransported from point A to point B under electroosmotic force, whilethe electrophoretic force is in one direction in one portion 142 and inthe opposite direction in the other portion 144.

To create a channel with sidewalls having positive or negative surfacecharges, one or both portions of the channel is coated with insulatingfilm materials with surface charges, such as a polymer. For example, inthe microfluidic system 100 the substrate 102 and the channels may beformed of glass. A portion of each channel is coated with a polymer ofopposite surface charge, such as polylysine, for example, or ischemically modified with a silanizing agent containing an aminofunction, such as aminopropyltrichlorosilane, for example. Furthermore,the surface charge densities and volumes of both channel portions shouldbe approximately the same to compensate for electrophoretic bias.

Rather than being formed in a solid planar substrate, the channel canalso be formed by two capillary tubes which are butted together with asalt bridge which separates an ionic fluid reservoir from fluids in thecapillary tubes. An electrode is also placed in the ionic fluidreservoir. One capillary tube has a negative surface charge and theother capillary tube a positive surface charge. The resulting capillarychannel operates as described above.

FIGS. 6A-6D illustrates another embodiment of the present invention forwhich the effects of electrophoretic bias induced upon the subjectmaterial is moving from point A to point B are compensated. In thisembodiment the subject material is mixed at point B, an intersectionbetween two channels, such as illustrated in FIG. 1.

FIGS. 6A-6D show a chamber 160 is formed at the intersection of thechannels 150, 152, 154 and 156. The chamber 160 has four sidewalls 162,164, 166 and 168. The sidewall 162 connects a sidewall of the channel152 to a sidewall of the channel 150; the sidewall 164 connects asidewall of the channel 154 to the other sidewall of the channel 152;the sidewall 166 connects a sidewall of the channel 156 to the othersidewall of the channel 154; and the sidewall 168 connects the oppositesidewall of the channel 156 to the opposite sidewall of the channel 150.Assuming a flow of materials through the channel 152 toward the channel156, the sidewalls 162 and 168 form as a funnel if the materials arediverted into the channel 150.

The dimensions of the sidewalls 162 and 168 accommodate the length of asubject material plug 200 traveling along the channel 152. The sidewalls162 and 168 funnel down the plug 200 into the width of the channel 150.The width of the channel 150 is such that diffusion of the subjectmaterial occurs across the width of the channel 150, i.e., mixing occursand any electrophoretic bias of the subject material created in thesubject material region 200 traveling along the channel 162 iseliminated. For example, if the channel 150 is 50 μm wide, diffusionacross the channel occurs in approximately one second for a moleculehaving a diffusion constant of 1×10⁻⁵ cm² /sec.

In FIG. 6A a plug 200 of cationic subject material is moving along thechannel 152 towards the channel 156. By the time the plug 200 reachesthe chamber 160, the subject material has undergone electrophoresis sothat the material is more concentrated at the forward end of the subjectmaterial region 200. This is illustrated by FIG. 6B. Then the voltagedrop impressed along the channels 152 and 156 is terminated and avoltage drop is created along the channels 154 and 150 to draw thesubject material region 200 into the channel 150. The sidewalls 162 and168 of the chamber 160 funnel the subject material region 200 with itselectrophoretically biased subject material. This is illustrated by FIG.6C. By diffusion the subject material is spread across the width of thechannel 150 before the subject material travels any significant distancealong the channel 150; the subject material in the subject materialregion 200 is mixed and ready for the next step of operation in themicrofluidic system 100.

In addition to its use in correcting electrophoretic bias within asingle sample, it will be appreciated that the structure shown in FIG. 6will be useful in mixing fluid elements within these microfluidicdevices, e.g., two distinct subject materials, buffers, reagents, etc.

EXAMPLES Example 1

Forced Co-migration of Differentially Charged Species, inElectropipettor-type Format

In order to demonstrate the efficacy of methods used to eliminate orreduce electrophoretic bias, two oppositely charged species wereelectrokinetically pumped in a capillary channel, and comigrated in asingle sample plug. A Beckman Capillary Electrophoresis system was usedto model the electrophoretic forces in a capillary channel.

In brief, a sample containing benzylamine and benzoic acid in either lowionic concentration (or "low salt") (5 mM borate), or high ionicconcentration ("high salt") (500 mM borate) buffer, pH 8.6, was used inthis experiment. The benzoic acid was present at approximately 2× theconcentration of the benzylamine. All injections were timed for 0.17minutes. Injection plug length was determined by the injection voltage,8 or 30 kV. The low salt and high salt buffers were as described above.

In a first experiment, three successive injections of sample in low saltbuffer were introduced into a capillary filled with low salt buffer.Injections were performed at 8 kV and they were spaced by low saltinjections at 30 kV. Data from these injections is shown in FIG. 7A.These data show that benzylamine (identifiable as short peaks, resultingfrom its lesser concentration) from the first and second injectionsprecede the benzoic acid peak (tall peak) from the first injection.Further, the benzylamine peak from the third injection is nearlycoincident with the first benzoic acid peak. Thus, this experimentillustrates the effects of electrophoretic bias, wherein sample peaksmay not exit a capillary channel in the same order they entered thechannel. As can be clearly seen, such separations can substantiallyinterfere with characterization of a single sample, or worse, compromisepreviously or subsequently introduced samples.

In a second experiment, the capillary was filled with low salt buffer.Samples were injected by first introducing/injecting high salt bufferinto the capillary at 8 kV (first spacer region 1). This was followed byinjection of the sample in high salt buffer at 8 kV, which was followedby a second injection of high salt buffer at 8 kV (first spacer region2). Three samples were injected in this manner, and spaced apart byinjecting a low salt buffer at 30 kV. As can be seen in FIG. 7B, bothcompounds contained in the sample were forced to co-migrate through thecapillary channel in the same sample plug, and are represented by asingle peak from each injection. This demonstrates sample registration,irrespective of electrophoretic mobility.

By reducing the size of the low salt spacer plug between the samples,relative to the size of the samples, partial resolution of thecomponents of each sample injection can be accomplished. This may beuseful where some separation of a sample is desired duringelectrokinetic pumping, but without compromising subsequently orpreviously injected samples. This was carried out by injecting thelow-salt spacer plug at 8 kV rather than 30 kV. The data from thisexample is shown in FIG. 7C.

Example 2

Migration of Subject Materials through Electropipettor into MicrofluidicSystem Substrate

FIGS. 9A-9C illustrate the experimental test results of the introductionof a subject material, i.e., a sample, into a microfluidic systemsubstrate through an electropipettor as described above. The sample isrhodamine B in a phosphate buffered saline solution, pH 7.4. A highionic concentration ("high salt") buffer was also formed from thephosphate buffered saline solution, pH 7.4. A low ionic concentration("low salt") buffer was formed from 5 mM Na borate, pH 8.6, solution.

In the tests subject material regions containing the fluorescentrhodamine B were periodically injected into the capillary channel of anelectropipettor which is joined to a microfluidic system substrate. Highsalt and low salt buffers were also injected between the subjectmaterial regions, as described previously. FIG. 9A is a plot of thefluorescence intensity of the rhodamine B monitored at a point along thecapillary channel near its junction with a channel of the substrateversus time. (In passing, it should be observed that the numbers onfluorescent intensity axis of the FIGS. 9A-9C and 10 plots are forpurposes of comparative reference, rather than absolute values.) Theinjection cycle time was 7 seconds and the electric field to move thesubject material regions through the electropipettor was 1000 volts/cm.The integration time for the photodiode monitoring light from thecapillary channel was set at 10 msec. From the FIG. 9A plot, it shouldbe readily evident that light intensity spikes appear at 7 secondintervals, matching the injection cycle time of the fluorescentrhodamine B.

In another experiment, the same buffers were used with the rhodamine Bsamples. The monitoring point was in the channel of the substrateconnected to the electropipettor. The injection cycle time was set at13.1 seconds and the voltage between the source reservoir containing therhodamine B and destination reservoir in the substrate set at -3000volts. The monitoring photodiode integration time was 400 msec. As shownin FIG. 9B, the fluorescent intensity spikes closely match the rhodamineB injection cycle time.

The results of a third experimental test are illustrated in FIG. 9C. Inthis experiment the electropipettor was formed from a substrate andshaped by air abrasion. The monitoring point is along the capillarychannel formed in the substrate (and a planar cover). Here the samplematerial is 100 μM of rhodamine B in a buffer of PBS, pH 7.4. The highsalt buffer solution of PBS, pH 7.4, and a low salt buffer solution of 5mM Na borate, pH 8.6, are also used. Again periodic spikes offluorescent intensity match the cyclical injection of rhodamine B intothe electropipettor.

FIG. 10 illustrates the results of the cyclical injection of anothersubject material into an electropipettor, according to the presentinvention. In this experiment the sample was a small molecule compoundof 100 μM with 1% DMSO in a phosphate buffered saline solution, pH 7.4.A high salt buffer of the same phosphate buffered saline solution, pH7.4, and a low salt buffer of 5 mM Na borate, pH 8.6, was also used. Theapplied voltage to move the subject material regions through theelectropipettor was -4000 volts, and the integration time for thephotodiode monitoring light from the capillary channel was set at 400msec. The samples were periodically injected into the electropipettor asdescribed previously. As in the previous results, the FIG. 10 plotillustrates that for small molecule compounds, the electropipettor movesthe samples at evenly spaced time (and space) intervals.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques described above may be usedin various combinations. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed is:
 1. A microfluidic system with compensation forelectrophoretic bias, comprising:a first capillary channel; a secondcapillary channel intersecting said first capillary channel; and achamber at said intersection of said first and second capillary channelsshaped such that a region containing subject material moving from saidfirst capillary channel to said second capillary channel is mixed tocompensate for electrophoretic bias in moving along said first capillarychannel; wherein said chamber is defined by sides along a firstcapillary channel length funneling into said second capillary channel.2. The microfluidic system of claim 1 wherein said chamber sides arestraight.
 3. The microfluidic system of claim 1 wherein said secondcapillary channel width is approximately equal to a length of saidsubject material region.
 4. The microfluidic system of claim 1 whereinsaid subject material has a diffusion constant and said second capillarychannel has a width such that said subject material diffuses across saidsecond channel width before being diverted from said second channel. 5.The microfluidic system of claim 4 wherein said subject material has adiffusion constant of approximately 1×10⁻⁵ cm² /sec. and said secondchannel width is approximately 10 μm.
 6. A microfluidic system,comprising:a body structure; a first microscale channel disposed in thebody structure; a second microscale channel disposed in the bodystructure, the second channel intersecting the first channel at a firstintersection; a chamber disposed in the body structure at theintersection, side walls of the chamber funneling into the secondchannel; and an electrokinetic material transport system fortransporting a subject material region from at least one of the firstchannel into the second channel through the chamber.
 7. The microfluidicsystem of claim 6, wherein the chamber is defined by sides along thefirst channel length funneling into the second microscale channel. 8.The microfluidic system of claim 7, wherein the sides of the chamber arestraight.
 9. The microfluidic system of claim 6, further comprising atleast a first subject material disposed in and being transported alongthe first microscale channel.
 10. The microfluidic system of claim 9,wherein the second microscale channel width is approximately equal to alength of the subject material region.
 11. The microfluidic system ofclaim 9, wherein the subject material has a diffusion constant and thesecond channel has a width, whereby the subject material diffuses acrossthe second channel width while the subject material region is in thechamber.
 12. The microfluidic system of claim 11, wherein the subjectmaterial has a diffusion constant of approximately 1×10⁻⁵ cm² /sec andthe channel width is approximately 10 μm.
 13. The microfluidic system ofclaim 6, wherein the body structure comprises:a first planar substratehaving a first planar surface, the first and second microscale channeland the chamber being fabricated into the first planar surface; a secondplanar substrate overlying the first planar surface of the firstsubstrate to cover the first and second microscale channels and thechamber.
 14. The microfluidic system of claim 13, wherein at least oneof first and second substrates is glass.
 15. The microfluidic system ofclaim 13, wherein at least one of the first and second planar substratesis a polymer.
 16. The microfluidic system of claim 6, wherein theelectrokinetic material transport system comprises at least first,second, and third electrodes, the first and second electrodes beingdisposed in electrical contact with the first channel on opposite sidesof the intersection, and the third electrode being disposed inelectrical contact with the second microscale channel, the first, secondand third electrodes being separately electrically coupled to a voltagesource for delivering a separate voltage to at least two of the first,second and third electrodes to produce a voltage gradient therebetween.17. A method of mixing a subject material region in a microfluidicsystem, comprising:providing a microfluidic device which comprises abody structure, a first microscale channel, a second microscale channelintersecting the first microscale channel and a chamber disposed in thebody structure at the intersection of the first and second channels, thechamber comprising side walls that funnel into the second channel;applying a first electric field along a length of the first channel tomove a first material region along the first channel and into thechamber, whereupon the first material region is mixed; and applying asecond electric field along a length of the second channel to move atleast a portion of the first material region from the chamber into thesecond channel.
 18. The method of claim 17, wherein the subject materialregion is electrophoretically biased during electrokinetic transportalong the first channel.
 19. The method of claim 17, wherein the firstsubject material has a diffusion constant and the second channel has awidth, such that the subject material diffuses across the width while inthe chamber.
 20. The method of claim 19, wherein the diffusion constantis approximately 1×10⁻⁵ cm² /sec and the width is approximately 10 μm.21. A method of electrokinetically transporting a first material regionthrough a microfluidic system while correcting for electrophoretic biasof the subject material in the subject material region,comprising:providing a microfluidic device having at least first andsecond microscale channels, the first and second microscale channelsintersecting at a first intersection, the first intersection beingdefined by a chamber that funnels into the second channel; transportingthe subject material along a first microscale channel into the chamber;and funneling the subject material from the chamber into the secondmicroscale channel, whereby the subject material diffuses across thesubject material region.